Cytotoxicity of haemolytic Listeria spp. in ciliates and amoebae was originally demonstrated by Chau Ly and Müller . They have shown that haemolytic L. monocytogenes and L. seeligeri induce lysis of T. pyriformis and Acanthamoeba castellani during 8-15 days while only few protozoa underwent lysis in the presence of non-haemolytic L. innocua.
Our results demonstrated that a L. monocytogenes mutant strain deficient in L. monocytogenes haemolysin, listeriolysin O (LLO) was incapable of impairing T. pyriformis growth compared to the isogenic wild type strain. A saprophytic species of L. innocua expressing LLO acquired toxicity in protozoa and caused their mortality and encystment. Thus, obtained results suggested that it is LLO that is responsible for L. monocytogenes cytotoxicity in protozoa.
Another observed LLO activity was stimulation of T. pyriformis encystment. Both cell death and encystment were responsible for decrease of trophozoite counts in the presence of L. monocytogenes. Here our results were in contradiction with previously published . Although cited above authors found that L. monocytogenes accelerates encystment of A. castellani, they did not observe T. pyriformis encystment independently on bacterial presence . This contradiction is related to the protozoan ability to encyst rather than LLO activity and might be due to different sources of a protozoan culture. Cyst formation by ciliates was described earlier  and cysts that we observed for the used T. pyriformis culture were similar to cysts depicted there (see Figure 1).
In contrast to wild type L. monocytogenes, LLO-expressing L. innocua caused a rapid decrease in counts not only trophozoites but as well cysts (see Figure 5). The constitutive LLO expression driven by PrfA* protein, which gene was inserted into the pHly/PrfA* plasmid, might be responsible for higher toxicity of L. innocua transformed with the plasmid. Wild type PrfA protein activity is regulated by co-factor binding, while the PrfA* protein is locked in the active conformation by a Gly145Ser substitution . Obtained results suggested that PrfA activity and LLO expression by intracellular L. monocytogenes might be switch off after host cell encystment but this is not possible for PrfA* protein. Further studies with using L. monocytogenes prfA*  are needed to get evidences in support of this suggestion.
Another pathogenic bacterium, a common representative of natural ecosystems, L. pneumophila was demonstrated to be cytotoxic for amoeba and to kill A. polyphaga via induction of necrosis due to Legionella pneumophila pore-forming activity . A similar mechanism might be responsible for the cytotoxic effect of LLO. LLO belongs to the family of cholesterol-dependent haemolysins, which includes streptolysin O and pneumolysin O [13, 14]. Proteins of this family can form oligomeric rings that plunge into membrane and generate pores . Therefore, LLO pore-forming activity might be responsible for L. monocytogenes cytoxicity in protozoa.
Our observations on the reduced growth of the hly gene deficient mutant in the co-culture with T. pyriformis compared to isogenic wild type bacteria are in line with a previous report that a hly gene deletion prevented L. monocytogenes from A. castellanii phagosome escaping . Phagosome escaping is prerequisite for L. monocytogenes replication in mammalian but not insect cells . It is not clear at present how the failure to escape the phagosome impairs intracellular growth in protozoan cells. However, the improved intracellular survival in synergy with rapid reduction of trophozoite concentration might be responsible for the advantages that LLO exerts on bacterial survival in the presence of actively grazing protozoa.
Considering the natural environment, LLO production seems to increase L. monocytogenes survival compared to non-haemolytic bacteria. Obtained results demonstrated higher counts for wild type L. monocytogenes than for the isogenic LLO deficient mutant during first days of co-cultivation supposing that wild type bacteria better survived upon initial interactions with the predator than non-haemolytic counterparts. Furthermore, prolonged bacterial survival might be supported by bacterial maintenance in protozoan cysts forming due to LLO activity. It is generally accepted that entrapped bacteria may benefit from the protective coat conferred by protozoan [28–30]. It has been demonstrated previously that encysted bacteria could survive sewage water treatment, which is fatal to free living bacteria . Survival of human pathogens inside protozoan cysts was demonstrated previously for Vibrio cholerae, L. pneumophila, Mycobacterium spp and an avirulent strain of Yersinia pestis [32–34]. However, to our knowledge active stimulation of protozoan encystment by bacteria was demonstrated only in case of L. monocytogenes (; and this work). Maintenance of pathogenic bacteria within cysts not only protects them from unfavorable environmental conditions but as well can preserve at the first stages of interactions with the macroorganism. That might be an important mechanism for bacterial spreading in the natural ecosystems when cyst protection not only supports pathogen survival in the hostile environment but as well increases its chance to multiply upon host invasion.
Involvement of LLO in different aspects of interactions between L. monocytogenes and protozoa has a striking similarity with its multiples roles during infection in mammals. Phagosome membrane disruption is the major role for LLO in intracellular parasitism in mammalian cells [2, 14]. However, LLO input in L. monocytogenes virulence is not limited to phagosome escaping: LLO generates a calcium flux into cells, promotes bacterial invasion in certain epithelial cells, and causes apoptosis in dendritic cells and T lymphocytes [13, 17, 18]. In protozoa, LLO is required for phagosome escaping likewise it takes place in mammalian cells ; it exerts a cytotoxic effect on protozoa (; and this work); at last, our results suggested that LLO causes protozoan encystment. Possible parallels between LLO-mediated mechanisms causing apoptosis in immune cells and encystment in protozoa require a special investigation.
Despite the growing number of evidences that a prey-predator model describing interactions between protists and saprophytic bacteria, is not appropriate to explain the interactions of bacteriovorous protozoa and pathogenic bacteria, the mechanisms that permit pathogenic bacteria to avoid protozoan grazing are not clear. It was suggested that these mechanisms may involve at least in part the means that pathogens utilize to survive in higher eukaryotes [28–30, 35]. Moreover, it was suggested that the resistance to digestion by bacteriovorous protozoa might be an evolutionary precursor of bacterial adaptation to intracellular survival in mammalian professional phagocytes such as macrophages. Our results support this hypothesis by demonstration of the role that the major virulence factor listeriolysin O (LLO) plays in interpopulation relationships of the pathogenic bacterium L. monocytogenes and the bacteriovorous ciliate T. pyriformis.
Discussing the input of LLO in interactions of L. monocytogenes with mammals and protozoa, it is necessary to take notice of LLO expression under different conditions. Expression of the PrfA protein, which is a master-regulator of virulence genes in L. monocytogenes , changes in a temperature-sensitive manner that results in very low expression of PrfA-controlled genes under environmental temperatures while their expression increases at the temperatures of mammalian body . In contrast to other virulence factors, the LLO-encoding hly gene expression is regulated by both PrfA-dependent and PrfA-independent promoters . Low LLO expression at environmental conditions driven by the PrfA-independent promoter and the low-active PrfA-dependent promoter is sufficient to provide L. monocytogenes with benefits in its interactions with other members of the natural ecosystems. Increasing LLO expression, e.g. via introduction of the PrfA* protein, which stimulates higher expression from the PrfA-dependent promoter, distorts the balance causing mortality not only among trophozoites but as well among cysts as we observed for L. innocua carrying pHly/PrfA* plasmid. Therefore, mutations resulting in increased LLO production might be detrimental for survival in the nature. It is interesting, that another Listeria virulent species, L. ivanovii, which is highly haemolytic and is not able to repress virulence factor production via a described PrfA-dependent mechanism , is much more rear isolated from environment than L. monocytogenes [39, 40]. Thus, LLO expression might be beneficial under different conditions but it is required a tight regulation in dependence on external conditions.